This research is designed to develop a new tool for measuring functional neural activity from the sub-cellular to the system scale. Using two-photon microscopy of the NADH/FP signal in vitro in cultured PC12 cells and in vivo in zebrafish, we will establish the important neuronal, activation-related metabolic events that cause modulations in the fluorimetric signal. In cultured PC12 cells, we will establish the spatio-temporal signature of known cellular physiological events such as stretch, pressure change, action potential generation and calcium spikes in benchmark, two-photon imaging studies giving important information on the relative amplitudes and durations of the metabolic events that we will use as proxies for the measurement of neuronal electrical and signaling activity. Using zebrafish, we will bring our imaging effort to the organismal level. We will measure intrinsic NADH/FP signal modulation in the well-characterized trigeminal ganglion. By comparing the NADH/FP signal with standard indicators of neuronal activity, we will characterize the spatio-temporal NADH/FP signal in a live preparation determining the detailed dynamics of the signal and determining differences with the signal arising from cultured cells. Because of the relatively low fluorescence amplitudes of NADH and FP relative to extrinsic fluorophores, the signal-to-noise ratio in two-photon NADH/FP imaging measurements limits the accuracy of quantitative measurements. Therefore, we will develop multivariate statistical analysis methods for estimating the multichannel NADH and FP signals. This will allow us to more accurately detect and quantitate metabolic changes in intact tissue. The resulting methods will be generally applicable to other dual-channel, spectroscopic signals. [unreadable] [unreadable] [unreadable]